PROJECT SUMMARY Phosphosignaling coordinates much of the host macrophage?s (M??s) response to infection with Mycobacterium tuberculosis (Mtb). Surprisingly, only few studies to date have measured global changes in the phosphoproteome upon infection with Mtb by mass spectrometry, and none in primary human M?s. In addition, a major challenge in the cell signaling field today is to separate phosphorylation events that are functional from those that are not. The lack of tools, experimental or computational, to d istinguish functional from non-functional phosphorylation events is the reason why many current studies of phosphosignaling largely remain inventories of phosphosites that provide limited biological insight. Combining phospho- and structural proteomics, we will test the hypothesis that the occurrence of a structural change of a protein upon phosphorylation is a reliable predictor of functional phosphorylation events. We will test this idea by combining detailed phosphosite mapping and global protein structure measurements to compare the proteomes of uninfected to Mtb-infected primary human M?s. We will correlate changes in individual phosphorylation sites with changes in the proteolytic cleavage pattern as a readout of structural change. Phosphorylation events that change a protein?s structure will be tested for their function in Mtb infection. We will knock out (or down) the phosphoprotein and test the effect on bacterial loads and M? function. To link effects of knockout to specific phosphorylation sites, we will complement with the protein bearing phosphoablative and -mimetic mutations and test for effects on M? signaling and Mtb infection. This project will provide a proof-of-principle for an experimental method to identify functional phosphorylation sites in general, and in particular in the context of the Mtb-infected M?. These data will allow for more meaningful curation of M? phosphorylation datasets, provide a global, functional view of the cell?s remodeling upon infection, highlight the specific immune pathways triggered by Mtb infection, identify potential new host-directed therapy targets, and identify downstream effectors that control or exacerbate Mtb infection.